Our current Merit Award focuses on factors contributing to virulence in Clostridium difficile, and a number of surface-layer proteins (SLPs) have been identified, that mediate bacterial adhesion to host intestinal epithelial cells. We now propose to significantly expand on these studies with the ultimate goal of translating our findings to a safe, easily utilizable, probiotic-based intervention for prevention of C. difficile infection (CDI). Three Specific Aims are proposed. First, we will define mechanistic bases of CD adherence to host epithelia mediated by surface-layer proteins (SLPs). CD mutagenesis and animal models of CDI will be exploited for these studies. Second, we will characterize non-SLP factors involved in CD colonization. Both bacterial and host- response proteins involved in, and contributing to, CD colonization as well as colonization resistance will be studied. Third, we will translate our bench-research findings to a clinically relevant outcome by developing a new adherence-based CDI intervention. This will involve construction of a targeted CD colonization inhibitor, by engineering a probiotic bacterium (Lactobacillus acidophilus) to express the CD colonization protein SlpA. We will validate the probiotic developed above by testing it against frequently isolated single-episode and relapse strains of CD recovered from surveillance studies performed at our VA hospital. For all studies in this proposal, we will use both the hamster and mouse models of acute CDI and CD colonization. We will also incorporate state-of-the-art methodologies including automated mass spectrometry and live-animal bioluminescence imaging to track the fate of ingested CD spores. PUBLIC HEALTH RELEVANCE: Over 400,000 cases of Clostridium difficile infection (CDI) occur annually in the USA, imposing a burden of >$3 billion on the healthcare system. Risk factors for CDI include hospitalization, age >65, co-morbid conditions, and antibiotic/PPI use - all of which are immediately relevant to patients treated in VA hospitals. Upto 11% of veterans also relapse with multiple recurrences of the disease. Severity of initial infection as well as relapses are also much higher with the newly emerged "hypervirulent" strains of C. difficile that are now common in VA hospitals. The studies proposed in this application are focused on understanding how C. difficile colonizes the human gut, and to validate a safe, cost-effective, easily tolerated intervention to prevent this establishment. The proposed work is thus consistent with the Merit Review mechanism, and is directly relevant to Veterans health.